Dopamine (DA) uptake by the DA transporter (DAT) and DAT cell surface expression is regulated by DAT substrates, inhibitors, and protein tyrosine kinases. However, DAT regulation by these biochemical mechanisms is not entirely understood, especially in specific brain areas. For instance, little is known about the relationship between DAT substrates/inhibitors and tyrosine kinases in regulating DAT function in the nucleus accumbens (NAc) and dorsal striatum (STR). To account for known differences in DAT activity and expression in these two regions, the use of brain preparations is essential for the study of DAT functional regulation. Therefore, the proposed experiments will examine [3H]DA uptake and DAT cell surface expression after exposure to DAT substrates (e.g., amphetamine) and inhibitors (e.g., cocaine) and tyrosine kinase activators and inhibitors, both alone and in combination with each other, in 1) NAc and STR synaptosomes and 2) midbrain-NAc and midbrain-STR organotypic slice co-cultures. Regulation of DAT activity and expression in the co-cultures will first be characterized after basal and stimulus-evoked DA release. Use of these two brain preparations will allow comparison of novel findings with previous research and assessment of the viability of slice cocultures as a physiological model for the study of DAT functional regulation in neurons. These studies will help elucidate the biochemical mechanisms underlying DAT regulation in brain under both physiological and drug-exposed conditions.